Abstract: Human GPR35 (hGPR35), a recently deorphanized Class A G-protein coupled receptor, has been shown to exhibit prominent expression in immune and gastrointestinal tissues, with additional expression in pancreatic islets, skeletal muscle, lung tissue, and the dorsal root ganglion. The rat GPR35 (rGPR35) analog, which has 72% sequence identity with human GPR35, has been shown to have expression in similar tissues as with human GPR35. GPR35 has been suggested to be involved in metabolism, heart failure, inflammation, asthma, a mental retardation syndrome associated with the deletion on 2q37.3, type II diabetes, as well as gastric cancer formation, making GPR35 a potential target for the treatment of multiple diseases. Both zaprinast, the well characterized cGMP-PDE inhibitor, and pamoic acid, a compound which the FDA has classified as an inactive compound, act as agonists at GPR35. However, interesting species differences have been found with these agonists and key mutations have also revealed differences between these two ligands. Pamoic acid is considerably lower in potency in rat GPR35, while zaprinast has increased efficacy in rat GPR35. Further, mutation studies suggest an increase in the potency of zaprinast in a human GPR35 R6.58A mutation. Pamoic acid, on the other hand shows similar potency to wild-type in this same mutant. To probe the molecular origins of these differences, three separate homology models, an active (R*) hGPR35, an R* hGPR35 R6.58A(240) mutant, and an R* rGPR35 model, were constructed and docking studies were performed with the aforementioned ligands. These studies revealed that the change in residue 5.43 (P5.43 in human; S5.43 in rat) alters the shape of the binding pocket for pamoic acid. In addition, arginines which contribute significantly to the interaction of pamoic acid in hGPR35 (R6.58 and R7.32) become uncharged residues (Q6.58 and S7.32) in rat GPR35. The increase of the potency of zaprinast in the hGPR35 R6.58A mutant receptor is due to the loss of bulk at position 6.58 (R6.58(240)&die; A6.58(240)), that allows for additional interactions with the ligand. The statistically equivalent potencies of pamoic acid for the wild-type and R6.58A(240) mutant hGPR35 receptors is due to the isoenergetic interchange of the direct interaction residue R6.58(240) with R7.32(255) in the R6.58(240)A mutant.